Causes, Effects and Molecular Mechanisms of Testicular Heat Stress

ARTICLE IN PRESS Reproductive BioMedicine Online (2014) ■■, ■■–■■

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REVIEW Causes, effects and molecular mechanisms of testicular heat stress

Damayanthi Durairajanayagam, Ashok Agarwal *, Chloe Ong

Center for Reproductive Medicine, Glickman Urological and Kidney Institute, Cleveland Clinic, Cleveland, Ohio, USA * Corresponding author. E-mail address: [email protected] (A Agarwal).

Damayanthi Durairajanayagam, PhD is a Senior Lecturer in Physiology at the Faculty of Medicine, MARA Univer- sity of Technology (UiTM), Malaysia. She is a past recipient of the Fulbright Research Exchange Scholar Award and recently completed her Research Fellowship at the Center for Reproductive Medicine, Cleveland Clinic, USA. Her research interests include oxidative stress, antioxidants and male infertility, and the use of proteomics and bioinformatics in studying the molecular markers of oxidative stress in infertile males.

Abstract The process of spermatogenesis is temperature-dependent and occurs optimally at temperatures slightly lower than that of the body. Adequate thermoregulation is imperative to maintain testicular temperatures at levels lower than that of the body core. Raised testicular temperature has a detrimental effect on mammalian spermatogenesis and the resultant spermatozoa. Therefore, thermoregulatory failure leading to heat stress can compromise sperm quality and increase the risk of infertility. In this paper, several different types of external and internal factors that may contribute towards testicular heat stress are reviewed. The effects of heat stress on the process of spermatogenesis, the resultant epididymal spermatozoa and on germ cells, and the consequent changes in the testis are elaborated upon. We also discuss the molecular response of germ cells to heat exposure and the possible mechanisms involved in heat-induced germ cell damage, including apoptosis, DNA damage and autophagy. Further, the intrinsic and extrinsic pathways that are involved in the intricate mechanism of germ cell apoptosis are explained. Ultimately, these complex mechanisms of apoptosis lead to germ cell death. © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

KEYWORDS: germ cell apoptosis, male infertility, molecular mechanisms, risk factors, scrotal hyperthermia, sperm DNA damage

Introduction (Zhu et al., 2004). Spermatozoa resulting from sperm cells exposed to hyperthermia in mice undergo apoptosis (Yin et al., 1997b) and contain damaged DNA (Perez-Crespo et al., 2008), The lack of thermoregulation of scrotal temperature causes leading to poor fertilizing capacity in vivo and in vitro (Yaeram testicular hyperthermia, which leads to genital heat stress. et al., 2006). This is detrimental to spermatogenesis and results in sper- Signiﬁcant apoptotic loss of germ cells after testicular heat matozoa of inferior quality. Both the epididymal sperm and stress may occur either through intrinsic or extrinsic path- testicular germ cells are sensitive to damage by heat stress ways. The molecular events that arise in germ cells exposed http://dx.doi.org/10.1016/j.rbmo.2014.09.018 1472-6483/© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Damayanthi Durairajanayagam, Ashok Agarwal, Chloe Ong, Causes, effects and molecular mechanisms of testicular heat stress, Repro- ductive BioMedicine Online (2014), doi: 10.1016/j.rbmo.2014.09.018 ARTICLE IN PRESS 2 D Durairajanayagam et al. to heat stress include the pro-apoptotic Bax and anti-apoptotic testis, followed by spermiogenesis in the epididymis. Human Bcl-2, cytochrome C, caspases and other heat-induced factors spermatogenesis requires almost 74 days for a complete cycle, (Kim et al., 2013). The germ cell apoptosis response that whereas sperm cells complete epididymal maturation in about follows heat stress takes place in a developmental stage- 12 days. The sequential cellular events of the spermato- specific manner, with the spermatocytes (diplotene and pachy- genic process initiate at the basal compartment and con- tene) and spermatids being most prone to heat-induced clude at the apical compartment of the seminiferous tubules. changes (Lue et al., 1999; Setchell, 1998). The reason for this Testosterone plays a crucial role in maintaining normal sper- vulnerability, however, has not been elucidated. matogenesis at the seminiferous tubules. The severity of damage to sperm cells subjected to heat stress varies with the intensity, frequency and duration of heat Events at the basal compartment of the exposure (Collins and Lacy, 1969; Paul et al., 2008). When seminiferous tubules germ cell apoptosis occurs it is also influenced by the severity and duration of heat stress (Kim et al., 2013). The two main events that occur here are during spermato- In this review, the following are discussed: the effects of goniogenesis (type Apale spermatogonia renews itself to hyperthermia on spermatogenesis, the measurement methods generate the stem cell pool), and type Apale (spermato- of scrotal temperatures, the various modifiable and non- gonia develop into Type B spermatogonia), which pro- modifiable factors that could cause increased testicular tem- gress to preleptotene, followed by leptotene primary peratures, the molecular mechanism of apoptosis, DNA damage spermatocytes. and autophagy, changes in gene expression and the pathways of germ cell apoptosis in response to testicular heat stress. Events at the apical compartment of the seminiferous tubules Effects of heat stress on spermatogenesis The three main events that occur here are spermatocy- and testis togenesis (primary spermatocytes progress from zygotene, pachytene, diplotene stages, to secondary spermatocytes then Testicular thermoregulation haploid spermatids); early round spermatids develop into elon- gated spermatids and undergo spermiogenesis to form sper- For optimal spermatogenesis to occur, testicular tempera- matozoa with fully compacted chromatin; and spermiation tures are maintained 2–4°C lower than core body tempera- (maturation and subsequent release of spermatozoa into semi- ture (Mieusset and Bujan, 1995). The temperature within the niferous lumen). testes is reflected by the temperature of the surrounding scrotal sac. Thermoregulation of the testis is aided by several Spermiation and maturation in the epididymis characteristics of the scrotal sac, such as thin skin with minimal subcutaneous fat, dense sweat glands and scant hair distri- Once in the lumen, spermatozoa leave the testis through bution. The musculature and vasculature in the genitals play rete testis into the epididymis, where sperm cells increase a role in regulating testicular temperature as well. To maxi- in concentration (caput), undergo maturation (corpus), and mize heat loss, the cremaster muscle that surrounds the testes are stored (cauda). Sperm cells at the tail end of the epi- and spermatic cords and the dartos muscle that lies beneath didymis have achieved full maturation, fertilizing ability and the scrotal skin relax, causing the testes to hang away from motility. the abdomen and the scrotal skin to slacken, increasing the total surface area for easy heat dissipation. Further, vaso- Naturally occurring defects during spermatogenesis dilation of scrotal vessels and activation of sweat glands The spermatogenic potential for human reproduction is a mere promote heat loss when temperatures increase. 12%, as the remaining sperm cells that develop either de- The testis is also thermoregulated via the counter-current generate, undergo apoptosis or develop abnormally (Sharpe, mechanism. Testicular artery and veins facilitate heat ex- 1994). Defects may occur during any part of spermatogenic change from the ‘warmer’ inflowing arterial blood to the cooler process. In spermatogoniogenesis, failure of Type A sper- outgoing venous blood. This transfer of heat assures that pale matogonia to develop into Type B spermatogonia leads to sper- ‘cooler’ arterial blood reaches the testis while the ‘warmed’ matogenic arrest (Holstein et al., 1988). During a defective venous blood disperses heat through the thin, scrotal skin ( Glad meiotic phase, apoptotic spermatocytes and spermatogenic Sorensen et al., 1991). Testicular veins carrying the ‘warm’ arrest of primary spermatocytes may occur. The cytoplasm blood anastomose and drain into the pampiniform plexus. In (cytoplasmic droplet) of the immature sperm cell is elimi- the case of varicocele, the pampiniform plexus becomes nated in spermiation, however, in some defective sperma- dilated, causing stasis and backflow of ‘warm’ blood back into tozoa, the cytoplasm may still remain as excess residual the internal spermatic veins. The compromised counter- cytoplasm (Breucker et al., 1985). During spermiogenesis, when current heat exchange thereby contributes to the increased haploid spermatids transform into fully differentiated sper- testicular temperatures found in varicocele patients ( Setchell, matozoa, defects that may occur (which are likely attribut- 1998). able to genetic factors) include absence of the acrosome, absence of the midpiece of the flagellum and damaged nuclear Spermatogenesis condensation (Holstein et al., 2003). Defects in the morphology of the ejaculated spermatozoa (head, neck, midpiece, The developmental process of the male gamete involves sper- tail, or all) are commonly seen during analysis of the seminal matogenesis in the lumen of the seminiferous tubules in the fluid (WHO, 2010).

Spermatogenesis, however, may be interrupted partially to whole-body heat (37°C to 38°C × 8h× 3 consecutive days) or completely by factors such as oxidative stress, increased had similar sperm count but lower motility. These epididy- scrotal temperature, nutritional and hormonal imbalance, side- mal sperm also exhibited membranous changes, making these effects of therapeutic drugs and radiation, which will produce sperm more prone to apoptosis (Wechalekar et al., 2010). In defective spermatozoa. a previous study, when heat shock was applied to male mice at 42°C for 30 min, the resultant spermatozoa retrieved from the epididymis demonstrated reduced count, motility and vi- Effect of heat stress on sperm cells and the testis ability. Spermatozoa that resulted from heat-exposed spermatocytes and spermatids both had damaged DNA, with the Testicular thermoregulation is important to maintain testicu- spermatozoa from heat-exposed spermatids showing poorer lar temperature within an optimal range for spermatogen- DNA integrity (Perez-Crespo et al., 2008). esis. Increases in scrotal temperature, albeit within physiological range, negatively affect sperm quality (Hjollund Other changes in the testis et al., 2000). An increase of 1°C entails a 14% drop in sper- Shortly after heat exposure, a loss in testicular weight occurs. matogenesis, and consequently poorer sperm production (Wang This reduction in testicular weight can be ascribed to germ et al., 1997). The effect of temperature on male fertility is cell loss, mainly by apoptosis. Although testicular weight may evident with the mean scrotal temperature in infertile men be partially regained several weeks after heat exposure in the being higher than fertile men and the quality of sperm de- rat, the testis remains lighter than before (Setchell et al., teriorating further with higher increases in scrotal tempera- 2002). Microscopic examination of the rat testis showed mi- ture (Mieusset et al., 1987). tochondrial degeneration, dilatation of the smooth endoplas- Spermatogenesis, especially the differentiation and matu- mic reticulum, and wider spaces in both Sertoli and spermatid ration of spermatocytes and spermatids, is temperature- cells, after heat exposure (Kanter et al., 2013). The somatic sensitive. Spermatogenesis should occur ideally at a minimum cells of the rat testis such as the Sertoli cells (Cai et al., 2011) of 2°C below core body temperature (Chowdhury and and Leydig cells (Kanter and Aktas, 2009) are also affected Steinberger, 1970; Thonneau et al., 1998). Elevated scrotal by heat stress, which renders them unable to provide a sup- temperature, however, causes testicular germinal atrophy, portive role to germ cell development. spermatogenic arrest (Munkelwitz and Gilbert, 1998) and decreased levels of inhibin B (a biochemical marker of spermatogenesis) (Jensen et al., 1997), which leads to lower sperm Measurement of scrotal temperature counts (Hjollund et al., 2002a).

The testis and epididymis represent the major thermal mass Effects of heat stress on germ cells in the hemiscrotum, and intrascrotal skin surface tempera- Germ cells are more vulnerable to heat stress as they have tures reﬂect the temperature of the underlying testis high mitotic activity (Shiraishi et al., 2012). Among the germ (Zorgniotti, 1991; Zorgniotti and Macleod, 1973). When mea- cells, the types that are most vulnerable to heat are the pachy- suring testicular and intra-scrotal temperatures, accuracy and tene and diplotene spermatocytes and the early round sper- reproducibility is essential as temperature differences in a eu- matids in humans (Carlsen et al., 2003) and rats (Chowdhury thermic and hyperthermic testis may be as little as 0.6– and Steinberger, 1970; Lue et al., 1999) alike. The basic 1.4°C (Zorgniotti and Macleod, 1973). Ideally, instruments used mechanisms with which germ cells incur damage include germ must be well-calibrated, have an accuracy of at least ±0.1°C, cell apoptosis (Lue et al., 1999, 2002; Yin et al., 1997b) and allow for fast and easy temperature measurement in differ- autophagy (Eisenberg-Lerner et al., 2009; Zhang et al., 2012), ent body positions as well as repeated measurements on the damaged DNA due to altered synapsis and strand breaks same area (Zorgniotti, 1982). Depending on the method of (Shikone et al., 1994; Yin et al., 1997b), and generation of measurement and presence or absence of pathology, testicu- reactive oxygen species (Ahotupa and Huhtaniemi, 1992; Ikeda lar temperature may range between 31.0°C to 36.0°C. et al., 1999; Peltola et al., 1995). The molecular responses Testicular temperature may be taken as single or continu- of germ cells of the hyperthermic testis will be further elabo- ous measurements. Single measurements of intrascrotal tem- rated in the section Molecular response of male germ cells perature are made using a mercury thermometer (clinical to heat stress. thermometers are inadequate for this purpose), skin surface thermocouples (for measurement in a clothed state, pro- Effects of heat stress on epididymal sperm vided the thermocouples stay in place), thermistor needles Epididymal sperm are affected by heat exposure differently (invasive method that directly measures intrascrotal tem- from germ cells. Male mice that were whole body-exposed peratures), infrared thermometry and thermography (a non- to temperatures of 36°C for 12 h on two successive days were contact method measuring scrotal skin heat emission or thermal found to have lower sperm count, lower testicular weight, radiation and not deep scrotal temperature) (Zorgniotti, 1982), less sperm fertilizing capacity in vivo and produced smaller and liquid crystal thermometry (Zorgniotti et al., 1982). The litter sizes compared with controls. Epididymal spermato- most widely used, repeatable method for single measure- zoa from the heated mice had lower sperm-zona pellucida ment is the invagination method using a mercury thermom- binding and oocyte penetration capacity. These effects were eter (Brindley, 1982; Zorgniotti and Macleod, 1973). In this ﬁrst seen at 1 week, and became more prominent at 2 weeks method, the patient is asked to disrobe from below the after heat exposure (Yaeram et al., 2006). In another study, waist and lay supine for equilibration to ambient room tem- sperm obtained from the cauda epididymis of mice exposed perature before measurement. Each testicle is measured

Please cite this article in press as: Damayanthi Durairajanayagam, Ashok Agarwal, Chloe Ong, Causes, effects and molecular mechanisms of testicular heat stress, Repro- ductive BioMedicine Online (2014), doi: 10.1016/j.rbmo.2014.09.018 ARTICLE IN PRESS 4 D Durairajanayagam et al. separately: a pre-warmed bulb of a mercury thermometer is compared with healthy men, attributing this to poor local ther- placed directly on the most prominent part of the anterior moregulation owing to physical movement constraints. Con- testis and the bulb held longitudinally against the scrotum. versely, Brackett et al. (1994) reported that scrotal Loose scrotal skin is drawn to envelope the thermometer bulb temperature was not the leading cause of abnormal sperm using the thumb and index finger. The expanded mercury quality (particularly sperm motility) in men with SCI, as men column will drop until it reached equilibrium, and the reading with SCI who walk (and do not use a wheelchair) also had poor at this point plus 0.1°C represents intrascrotal temperature. semen quality. Continuous measurements involve two cutaneous thermo- A predominantly sedentary or seated position during long couples or thermoprobes being taped to scrotal skin on the stretches of passive tasks such as working at the computer anterior face of each scrotum and connected to a small por- or commuting increases scrotal temperatures (Bujan et al., table data recorder attached to a belt worn by the patient. 2000; Hjollund et al., 2002b). The position of one’s legs and Measurements are recorded at 2-min intervals, allowing for the type of chair used also influence scrotal temperatures. continuous measurement of dynamic scrotal temperature re- For example, sitting cross-legged on a typical cushioned office cording (Bujan et al., 2000; Jockenhovel et al., 1990). For chair is likely to generate more scrotal heat compared with whole-day measurements, a thermistor is attached to the un- sitting on a saddle seat with wide-angled hips and knees, as derwear and is connected to a light-weight data logger the latter position promotes perigenital ventilation (Koskelo (Hjollund et al., 2002b). et al., 2005; Mieusset et al., 2007). Heat from the seated surface, as from a heated car seat or a heated floor, further adds to scrotal temperatures that are already elevated from Factors that contribute towards testicular being in a seated position (Jung et al., 2008a; Song and Seo, heat stress 2006). Layers of clothing and bedding trap additional layers of air and impede air exchange, thus conserving heat and increas- Maintaining a temperature difference between the body and ing scrotal temperatures. Being clothed elevates scrotal tem- testes is crucial to ensure the production of normal sperma- peratures by 1.5–2°C when standing or supine, compared with tozoa. In daily life, however, a multitude of external and in- an unclothed state (Mieusset et al., 2007; Zorgniotti et al., ternal factors could narrow this temperature difference, 1982). Thus, a choice of clothing that encourages good air flow thereby increasing the risk of abnormal spermatogenesis and could minimize the deviation of physiological scrotal tem- the changes associated with increased testicular heat expo- peratures. In this vein, the Scottish kilt (Kompanje, 2013) and sure. These thermogenic factors can be broadly grouped into the Asian sarong minus underwear would seem an ideal choice lifestyle and behavioural factors, occupational and environ- of leisure clothing, as the regular use of tight underwear was mental factors (external factors) and clinical factors result- found to reduce sperm quality (Laven et al., 1988; Lynch et al., ing from pathophysiological conditions (internal factors). 1986; Tiemessen et al., 1996). Although the effect of tight underwear versus boxer shorts on sperm parameters is in- conclusive, it would seem that tighter-fitting undergar- Lifestyle and behavioural factors ments would leave less room for scrotal movement and air circulation hence contributing to higher genital tempera- Lifestyle and behavioural causes of testicular heat stress en- tures (Munkelwitz and Gilbert, 1998). compass modifiable factors that are a result of habit or prac- tices that could be altered or avoided with conscious effort. Hot baths and sauna The use of hot baths and sauna for relaxation and rejuvena- Clothing and posture tion may make one feel better, but also has a negative effect Testicular temperature depends on scrotal position, which on semen quality. Full-body immersion in a warm bath, hot differs with postural changes. Scrotal temperature is at its tub, heated Jacuzzi or whirlpool at temperatures over 36.9°C lowest on an unclothed, upright body (Rock and Robinson, for 30 min or more a week for 3 months or more leads to 1965; Zorgniotti and Macleod, 1973) as the position of the un- wet hyperthermia, which could have a reversible negative covered, hanging testis allows for easy heat dissipation. Scrotal effect on sperm motility (Shefi et al., 2007). Users of typical temperatures are lower when walking compared with sitting saunas experience wet heat and warmed surfaces, whereas because scrotal movement during ambulation provides better modern infrared-type saunas offer dry, radiant heat. Studies air circulation and heat dispersion. Accordingly, testicular tem- show that, after sauna exposure, scrotal temperatures reach perature increases when movement is minimized, as in cases up to body temperatures within 10 mins, and there is a sig- of prolonged sitting (testis cradled between the thighs) or lying nificant but reversible negative effect on spermatogenesis down (scrotum resting on thighs) (Brindley, 1982; Rock and (Jockenhovel et al., 1990). In saunas with temperatures ranging Robinson, 1965). After at least 20 min of being seated, male from 80–90°C, and at different frequency and duration of paraplegics in wheel chairs (approximated, unmoving thighs) exposure, the use of saunas could disrupt spermatoge- had higher deep scrotal temperature and less motile sperm nesis and cause abnormal sperm count and motility compared with able-bodied men who sat freely (Brindley, (Brown-Woodman et al., 1984; Garolla et al., 2013; Saikhun 1982). Later studies differ in their results relating scrotal tem- et al., 1998). Further, regular sauna exposure over an entire perature with impaired sperm quality in individuals with spinal spermatogenic cycle also modified mitochondrial function, cord injury (SCI). Wang et al. (1992) reported a higher initial chromatin protamination and condensation in the sperm scrotal temperature in men in wheelchairs with SCI (Garolla et al., 2013).

Laptop use Professional or occupational drivers and individuals who In the current online information era, the use of laptops is have long daily commutes are more prone to having in- prevalent, and especially so in those within the reproduc- creased scrotal temperatures (Bujan et al., 2000), poorer tive age. As a computer on the lap, it is close to the genital sperm quality (Chia et al., 1994; Figa-Talamanca et al., 1996; area, and sitting with one’s legs close together for long hours Henderson et al., 1986; Sas and Szollosi, 1979) and longer time increases scrotal temperature, which may negatively affect to pregnancy (Thonneau et al., 1996). The negative effect of sperm parameters (Sheynkin et al., 2005). long hours of driving and seated commutes increases in severity with the number of years spent engaging in such ac- Cycling tivities (Figa-Talamanca et al., 1996; Sas and Szollosi, 1979). Among the different types of exercise, cycling is one that is Ambient heat reputed to impair male fertility. Aspects of cycling that may influence scrotal temperatures include posture, duration and A study on fertile, European men living in different cities re- intensity of cycling (Jung et al., 2008b), and the attire. Par- ported a general seasonal variation in sperm concentration ticularly in professional cyclists, extended periods of cycling and total sperm count, with summer values being approxi- in form-fitting spandex outfits and being seated for long hours mately 70% of their winter values. Their sperm motility and on a saddle seat is likely to cause elevated scrotal tempera- morphology, however, did not seem to vary with these seasons tures (Lucia et al., 1996). (Jorgensen et al., 2001). Other studies have shown a similar effect of changing seasonal temperature on sperm counts of Obesity healthy men (Gyllenborg et al., 1999; Tjoa et al., 1982), but this possible connection between seasonality and sperm con- Obesity is on the rise globally, and men with an above- centration was not seen in healthy Australian men (Mallidis normal body mass index (BMI) (≥25) have an average of 25% et al., 1991). lower sperm count and motility compared with men with a normal BMI (Kort et al., 2006). Not surprisingly, obesity rates tend to be higher in infertile men compared with men with Clinical factors euspermia (Hammoud et al., 2008). Obese men have compromised testicular thermoregulation owing to several factors: decreased physical activity and prolonged sedentary periods, Testicular hyperthermia resulting from pathological failure increased fat deposition in the abdominal, suprapubic, sper- of thermoregulation imposes adverse effects on the spermatic cord (scrotal lipomatosis) and upper thigh areas, which matogenic process. Abnormalities such as cryptorchidism and leads to suppressed spermatogenesis (Ivell, 2007; Shafik and varicocele result in exposure of the testis to raised tempera- Olfat, 1981a, 1981b). About 65% of infertile patients whose tures and compromised sperm quality, which may lead to a excess scrotal and suprapubic fat were removed showed im- loss in male fertility. provement in their sperm count, motility and morphology, with one in five of these patients successfully achieving a good preg- Cryptorchidism nancy outcome (Shafik and Olfat, 1981b). Failure of the testis to fully descend into the scrotal sac before birth or within the first few months of life could lead to subfertility and increased risk of testicular germ cell cancer Occupational and environmental factors (Toppari et al., 2014). An undescended testis is exposed to body temperatures and, although painless, it could lead to Job exposure to heat-generating conditions are significant con- heat-induced changes in the spermatogenic process, which tributors to heat stress, especially as work takes up a sub- affects the germ cells, spermatozoa, testis and testicular hor- stantial portion of the day and exposure to the source of mones (Bertolla et al., 2006; Lee and Coughlin, 2002; Li et al., radiant heat is likely to occur almost daily over long periods 2006; Liu and Li, 2010). The severity with which cryptorchi- of time. Another factor that may contribute to heat stress in dism affects fertility depends on whether one or both testes the male is ambient heat caused by hotter environmental tem- had failed to descend fully, its position along the inguinal peratures where the men live. canal, the cause of the incomplete descent and the length of time before surgical intervention to reposition the af- Radiant heat fected testicle into the scrotal sac (Agoulnik et al., 2012). Certain labour-intense jobs entail exposure to long periods of intense, radiant heat. Welders, for example, are exposed Varicocele to strong levels of heat, toxic metals and fumes during welding. Varicocele affects 15% of the male population, 40% of men Studies involving these workers demonstrate reversible decline with primary infertility and eight out of 10 men with second- in semen quality (Bonde, 1992; Kumar et al., 2003). Those ary infertility. It is the most commonly occurring and treat- working directly with sources of severe heat, such as bakers able cause of male infertility. Varicocele occurs when and ceramic oven operators, have a longer time to preg- abnormal dilatation of the veins of the pampiniform plexus nancy, which suggests that occupational heat exposure has that surrounds the vas deferens in the spermatic cord occurs, an effect on fertility (Figa-Talamanca et al., 1992; Thonneau which leads from the scrotum to the inguinal canal. The re- et al., 1997). Men who work in close range to sources of intense sulting backflow and stasis of blood hampers the counter heat, such as the rear end of a submarine (location of motor) current heat exchange between testicular arterial and venous seem to face infertility-related problems (Velez de la Calle blood, which consequently exposes the testis to tempera- et al., 2001). tures closer to the core body temperature (Setchell, 1998).

The temperature of a testis affected with varicocele is about 1997b). Apoptosis, also known as type I programmed cell 2.5°C higher than a normal testis (Goldstein and Eid, 1989) death, involves identifiable cellular changes, such as DNA frag- and in an infertile individual, scrotal hyperthermia is most mentation, cell volume shrinkage, and plasma membrane likely varicocele-related (Mieusset et al., 1987). Higher scrotal blebbing (Liu, 2010). During germ cell development, physi- temperatures in varicocele patients with infertility lead to ological germ cell apoptosis occurs to maintain the quality sperm DNA fragmentation and apoptosis, as well as hor- of the germ cell. Also, in the male germ cell, particularly, monal imbalance (Ku et al., 2005; Shiraishi et al., 2010). DNA damage is a precursor to apoptosis (Shaha et al., 2010). Henriksen et al. (1995) suggested that rat germ cells with heat- Febrile episodes damaged DNA were being eliminated via apoptosis. Using his- During the onset of a fever, testicular thermoregulation is dis- tochemical staining, Yin et al. (1997b) found evidence of rupted and scrotal temperature is raised along with the core apoptosis, especially among primary spermatocytes and round body temperature. Fever, which lasts for a day or longer, spermatids, and the effect was more noticeable in more affects the ongoing spermatogenic process. The magnitude mature rats. Lue et al. (1999, 2002) used the newer tech- of the temperature difference, duration (acute or pro- nologies of TdT (terminal deoxynucleotidyl transferase)- longed) and timing (which stage of the spermatogenic cycle) mediated dUDP nick-end labelling assay and electron of the febrile episode causes varying effects on the resul- microscopy to further confirm that rat and monkey germ cells tant spermatozoa (Carlsen et al., 2003; Evenson et al., 2000). were dying via apoptotic mechanisms. In addition to direct Higher temperature differences, longer periods of tempera- spermatozoa, DNA damage, heat may also denature cyto- ture dysregulation, or both, result in a more severe effect on plasmic bridges necessary for cell survival and affect fluid com- spermatogenesis. Further, particular stages of spermatogen- position in the cauda epididymis, which hinders proper esis seem more vulnerable to raised temperatures than others spermatozoa maturation, thus contributing to the increase (Carlsen et al., 2003). For example, fever for more than 1 day in apoptosis, both in rats and humans (Legare et al., 2004; that occurred when ejaculated sperm was undergoing sper- Rockett et al., 2001). This is known as the heat shock re- miogenesis (9–32 days before ejaculation) caused a de- sponse, which is facilitated by a group of proteins called the crease in sperm concentration (by 35%) and normal morphology heat shock proteins (HSP) (Izu et al., 2004; Widlak et al., 2007). (by 7.4%); as well as an increase in percentage immotile sperm There are two types of HSP: constitutive and inducible. (by 20.4%). Fever during mitotic proliferation (57–80 days Under normal circumstances, constitutively produced HSP are before ejaculation), meiosis (33–56 days before ejacula- molecular chaperones that ensure polypeptides are as- tion) and sperm maturation (up to 8 days before ejacula- sembled and transported correctly. They also contribute to tion) did not significantly affect these parameters, except for other cellular processes, such as stabilizing proteins in their reduced sperm concentration when fever occurred during inactive forms and inhibiting degradation (Neuer et al., 2000; meiosis (Carlsen et al., 2003). Son et al., 1999). On the other hand, as part of an innate pro- In summary, at any one time, many factors contribute to tective mechanism conserved through evolution, cells respond testicular temperatures: posture, clothing, type of activity, to heat stress by halting the synthesis of most proteins and occupational and environmental conditions as well as medi- diverting all resources available to produce inducible HSP cally related afflictions. For example, using a quilt over typical (Neuer et al., 2000; Pei et al., 2012; Widlak et al., 2007). These nightclothes while lying down in bed after a hot bath, or a proteins tend to oligomerize in order to carry out their func- patient with a varicocele sitting with legs close together in tions effectively (Sreedhar and Csermely, 2004). They protect well-fitting clothes on a couch with a laptop on the lap, will cells from heat stress by binding to proteins and preventing give a cumulative effect resulting in increased genital heat their denaturation and incorrect folding. The extent of in- of the individual. Depending on the circumstance, genital heat duction is dependent on the intensity and duration of heat exposure may be transient, prolonged or sustained with varying exposure – the higher the temperature and the longer the ex- degrees of intensity. The damaging effects of heat exposure posure, the greater the amount of HSP produced to protect on sperm parameters and male fertility tends to accumu- the cell. Because of its important function in ensuring correct late with repeated exposure over a period of time. assembly and transport of proteins, as well as protecting the cell against external stress, HSP are essential for spermatocytes to develop into healthy mature spermatozoa (Legare Molecular response of male germ cells to et al., 2004). heat stress Heat shock factor 1 (HSF1), a protein that is produced in- tracellularly in sperm cells when the HSF1 gene is activated by heat stress, has two paradoxical roles (Izu et al., 2004). Several studies (mainly using the cryptorchid model) have in- First, it is responsible for the rapid increase in HSP pro- vestigated the molecular aspects of male germ cell apopto- duced, thus promoting cell survival (Widlak et al., 2007). Con- sis after heat stress, and these findings are elaborated upon versely, it is also involved in directly eliminating aberrant germ in this section. cells to ensure that mature cells are of good quality (Izu et al., 2004; Widlak et al., 2007). The most common HSP activated Germ cell apoptosis by HSF1 is HSP70, which is found in elevated amounts in cryptorchid mice and rabbit testes (Pei et al., 2012; Rockett et al., Many experiments inducing cryptorchidism in animals have 2001). HSP70 not only acts as a regulator of p53, a tumour- shown an increase in apoptosis of germ cells, probably caused suppressor protein involved in extrinsic apoptosis, but is by DNA damage, resulting in a decline in testis weight and in- also involved in intrinsic apoptosis by causing an increase fertility problems (Banks et al., 2005; Setchell, 1998; Yin et al., in cytochrome C necessary for caspase (cysteine–aspartic

Please cite this article in press as: Damayanthi Durairajanayagam, Ashok Agarwal, Chloe Ong, Causes, effects and molecular mechanisms of testicular heat stress, Repro- ductive BioMedicine Online (2014), doi: 10.1016/j.rbmo.2014.09.018 ARTICLE IN PRESS Causes, effects and mechanisms of testicular heat stress 7 protease) activation (Sreedhar and Csermely, 2004; Widlak cell death. Autophagy is a process in which cells are phago- et al., 2007). Caspases are crucial mediators of apoptosis and cytosed by vesicles, degraded by lysosomes, and the result- can be divided into two groups: initiators and apicals and ex- ing cellular components recycled for energy generation ecutioners and effectors. Although the former are respon- (Eisenberg-Lerner et al., 2009; Zhang et al., 2012). When this sible for starting the apoptotic process, the latter are in charge pathway is activated by heat stress, the cytosolic form of light of carrying apoptosis out via cleavage of various proteins (Vera chain 3 (LC3B-I) is modiﬁed into a membrane-bound form et al., 2005). Moreover, HSP70 is purported to play a vital role (LC3B-II), and the ubiquitin-like conjugation system is acti- in the response to oxidative stress as well (Sreedhar and vated as well. The formation of LC3B-II assists in proper de- Csermely, 2004). These apoptotic and oxidative stress path- velopment of autophagosomes, and the autophagosome ways will be further elaborated upon in the section Molecu- formation by the conjugation system, both considered markers lar mechanism of heat stress – proposed pathways. of the autophagy process (Zhang et al., 2012). Blackshaw and Hamilton (1970) also argued that the rapid DNA degradation and change in acid phosphatase and amino-peptidase reac- Sperm DNA damage tions point to the involvement of lysosomes in the cellular response to heat stress in rats. Moreover, autophagy can work Despite the high rate of apoptosis, some cells are usually able in concert with apoptosis as well – either together or as a back- to survive cryptorchidism, but eventually develop into mature up mechanism when apoptosis fails (Eisenberg-Lerner et al., spermatozoa containing damaged DNA in murines (Banks et al., 2009; Zhang et al., 2012). 2005). This could partly be due to the protective effect of inducible HSP, which bind to proteins, thus preventing their denaturation and incorrect folding (Legare et al., 2004; Widlak Molecular mechanism of heat et al., 2007). Shikone et al. (1994) and Yin et al. (1997b) both stress – proposed pathways detected DNA fragmentation in cryptorchid mice testes, and this is likely to be the cause of infertility (Banks et al., 2005; The aforementioned responses occur through various diverse Shikone et al., 1994; Yin et al., 1997b). In rodents, chroma- pathways, and there may be some degree of crosstalk among tin structure was also found to be altered and chromatin ma- them too (Paul et al., 2009). The pathways that will be ex- terial reduced (Blackshaw and Hamilton, 1970; Sailer et al., plained in detail in this section are that of gene expression 1997). This loss of integrity is generally attributed to oxida- changes, stress response, impaired DNA repair as well as apop- tive stress, but could also be due to defective repair mecha- tosis (both intrinsic and extrinsic). nisms or destruction of the testes in murines such that spermatocytes are unable to develop properly (see section ‘Molecular mechanism of heat stress: proposed pathways’) Gene expression changes (Banks et al., 2005; Setchell, 1998). Increase in chromosomal abnormalities in the form of X-Y bivalent dissociations during metaphase I were also noted in Heat stress alters the levels of expression of various genes in experiments conducted by Garriott and Chrisman (1980) and a complex manner. It is still debatable, however, if the modu- van Zelst et al. (1995), in which male rats and mice were lation of gene expression levels are directly caused by heat exposed to hyperthermic conditions. This is because exces- stress or indirectly caused by other cellular changes (Setchell, sive heat prevents the synaptonemal complex from function- 1998). Together with post-translational modiﬁcation and ing normally, resulting in the formation of fewer and more protein localization, altered levels of gene expression will lead distal chiasma. Hence, bivalents are less strongly held to- to changes in the protein composition of the spermatocyte gether and the resulting presence of unpaired Y chromo- (Kim et al., 2013). Overall, the changes in gene expression, somes will cause spermatocytes to undergo apoptosis (Garriott as was seen in mice, indicate that the spermatocyteis un- and Chrisman, 1980). dergoing cellular shutdown in response to hyperthermia (up- In addition to damaging DNA, hyperthermia also causes a regulated genes) have defensive or regulatory roles that are decrease in DNA synthesis and the degradation of many mRNAs necessary for defense and repair, whereas all other genes are and proteins necessary for cell survival (Izu et al., 2004; down-regulated (Rockett et al., 2001). Nishimune and Komatsu, 1972; Tramontano et al., 2000). Acid In addition to the up-regulation of HSF1 gene coding for phosphatase and amino-peptidase reactions, considered es- HSF1 protein, which in turn increases HSP production, other sential for lysosomal function and normal protein synthesis genes that are up-regulated include those responsible for apop- respectively, were also altered in rats and the presence of tosis, cell adhesion and signal transduction. For instance, in multinucleated giant cells additionally caused a cumulative adult male mice, it was found that the expression of laminin decline in the number of viable mouse spermatozoa pro- and its receptors are up-regulated because laminin is reduced (Blackshaw and Hamilton, 1970; Waldbieser and quired to maintain Sertoli cell barrier functions vital for proper Chrisman, 1986). spermatogenesis (Rockett et al., 2001). In mice, the phoshoprotein p53, which is involved in cell cycle arrest and promotes apoptosis when DNA damage is too extensive to be repaired, is also up-regulated to detect damaged cells and Autophagy eliminate them (Absalan et al., 2012). Rockett et al. (2001) also highlighted that pro-apoptotic genes may be down- Finally, autophagy, commonly described as type II pro- regulated in mice after a few hours of heat stress in order to grammed cell death, could also be responsible for causing germ prevent complete destruction of all cells.

One of the genes that were found to be down-regulated, As for the second postulation, Ishii et al. (2005) argued that Bag-1, prevents normal HSP70 functioning such that the pro- the short duration of heat exposure (approximately 15 min), teins are not folded correctly. Thus, a decrease in Bag-1 would which caused damage to spermatocytes in SOD1-knockout result in increased activity of HSP70 (Rockett et al., 2001). mice, was an insufﬁcient amount of time for any ROS gener- Banks et al. (2005) showed that levels of cold inducible RNA ated to cause peroxidation and eventually apoptosis. Hence, binding protein (Cirp) are decreased, hence cellular pro- it was more likely that the ROS produced acted as a signal cesses like mitosis and meiosis are uncontrolled and germ cells to trigger apoptotic mechanisms. undergo apoptosis. Other genes such as DNA polymerase β and DNA ligase III, are down-regulated in male rats as well, preventing DNA repair from occurring and further contributing Impaired DNA repair pathway to apoptosis (Tramontano et al., 2000).

The effects of hyperthermia could also occur as a result of impaired DNA repair mechanisms. As gene expression levels Stress response pathway of DNA polymerase beta and DNA ligase III levels reduce in response to heat exposure, the germ cell’s ability to repair Free radicals are molecules with at least one unpaired elec- its DNA decreases (Tramontano et al., 2000). Additionally, tron, making them extremely unstable and highly reactive poly(ADP) ribose polymerase, another enzyme involved in DNA (Sharma and Agarwal, 1996). They collide into neighbouring repair, is substantially decreased in cryptorchid rat testes molecules and cause propagative chain reactions that produce (Tramontano et al., 2000). Banks et al. (2005), however, pro- even more free radicals (Tremellen, 2008). Reactive oxygen posed an alternative theory, which states that DNA damage species (ROS) such as superoxide anions, hydroxyl radicals, caused by excessive heat could be too great for the natural and hypochlorite radicals, are produced during oxygen me- testicular repair mechanisms to cope with and thus, some but tabolism and are found in the testes because they help in sper- not all, of the DNA damage is repaired. As such, although still matozoal functions such as capacitation, acrosome reaction, damaged, the DNA may persist into mature, motile sperma- hyperactivation, and sperm-oocyte fusion (Agarwal et al., tozoa. Thus, in an assisted reproduction technique setting, 2012; Shiraishi et al., 2012). To maintain ROS at an accept- particularly in intracytoplasmic sperm injection cycles, sper- able level, natural antioxidants, such as vitamins C and E and matozoa selection should take into account the DNA integ- carotenoids are present in the testes. When this balance of rity and its role in spermatozoa viability (Banks et al., 2005). free radicals and antioxidants is upset, oxidative stress occurs and this results in apoptosis (Agarwal et al., 2012; Paul et al., 2009). Apoptotic pathways Reactive oxygen species are known to be produced in cryptorchid testes and testes with varicocele, and there are two Heat stress results in the above mechanisms – gene expres- probable ways in which they could be involved in the heat sion changes, stress response and impaired DNA repair – which stress response. Firstly, oxidation of cellular components such eventually lead to apoptosis of abnormal germ cells. Apop- as DNA and lipids could lead to apoptosis directly and, sec- tosis can occur intrinsically or extrinsically (Figure 1). ondly, the generation of ROS could indirectly trigger the activation of apoptosis (Ishii et al., 2005; Shiraishi et al., 2010). To support the ﬁrst hypothesis of directly causing apop- Intrinsic apoptotic pathway tosis, Ahotupa and Huhtaniemi (1992) and Ikeda et al. (1999) The intrinsic apoptotic pathway, also known as the found that when rats’ testes were exposed to heat stress, an mitochondria-dependent apoptotic pathway, occurs in all cells increase in hydrogen peroxide and, hence, lipid peroxidation and is triggered by the translocation of certain members of was accompanied by a decrease in the activity of enzymatic the Bcl-2 protein family, such as the pro-apoptotic Bax (Bcl- antioxidants such as superoxide dismutase and catalase. As 2-associated X protein) and anti-apoptotic Bcl-2 (B-cell lym- such, compromised antioxidant capabilities led to higher ROS phoma 2) (Liu, 2010). In healthy cells, Bax is largely found concentrations and more oxidative stress. In fact, Ikeda et al. in the cytosol. In response to heat stress, however, Bax ac- (1999) further demonstrated that treating the rats with cata- cumulates in the mitochondria and endoplasmic reticulum, lase reduced the amount of peroxidation and apoptotic cells, whereas Bcl-2 localizes on the mitochondrial membrane (Hikim therefore lending even more support to the hypothesis. The et al., 2003; Liu, 2010; Vera et al., 2004). The relocation of results of another experiment conducted by Peltola et al. Bax is also coupled with the concentration of ultra-condensed (1995) in rats, however, suggested that the increased oxida- mitochondria in paranuclear areas of spermatocytes des- tive stress was more likely a result of the rapid increase in tined for apoptosis (Liu, 2010; Vera et al., 2004). Although ROS rather than the decreased antioxidant capacity. This is Bcl-2 is phosphorylated and thus inactivated, Bax is in- because the observed decline in manganese superoxide serted into the outer mitochondrial membrane, which leads dismutase was not consistent with the decrease in amount of to conformational changes that allow the release of cyto- normal DNA. This indicates that the decrease in antioxi- chrome C, into the cytosol (Hikim et al., 2003; Kim et al., dants was not the main factor causing increased DNA damage 2013). Cytochrome C is a small protein that plays a major role and that the increase in ROS most likely had a more signiﬁ- in the electron transport chain, and is commonly found on the cant role. Regardless of the cause of increased oxidative stress, inner mitochondrial membrane taking part in redox reac- the peroxidation of cellular components is still likely to be tions. In the cytosol, cytochrome C interacts with apoptotic involved in apoptosis. protease activating factor 1 (Apaf-1) to form a complex (Hikim

Figure 1 Intrinsic and extrinsic pathways of apoptosis. The intrinsic apoptotic pathway is dependent on the mitochondria: Bax (a pro-apoptotic gene) responds to heat stress and accumulates in the mitochondria, whereas Bcl-2 (an anti-apoptotic gene) localizes on the mitochondrial membrane. Bcl-2 is phosphorylated and becomes inactive. Bax is inserted into the outer mitochondrial membrane leading to conformational changes that allow the release of cytochrome C into the cytosol. Cytochrome C interacts with Apaf-1 (apoptotic protease activating factor 1) to form a complex. Activated Apaf-1 binds to caspase 9 and proteolytically activates the caspase cascade via executioner caspases 3, 6 and 7. The extrinsic/death receptor apoptotic pathway: this pathway is initiated upon heat stress exposure, when death receptor Fas ligates to its ligand FasL, causing Fas to recruit FADD (Fas-associated death domain) through shared death domains (DD). When the Fas/FADD complex binds to initiator caspases 8 or 10 with its N-terminal DED (death effector domain), activated DD trigger the caspase cascade, leading to its activation via executioner caspases 3, 6 and 7. Also during hyperthermia, p53 (a tumour suppressor that increases the expression of pro-apoptotic genes) is relocated from the nuclear envelope to the nucleus, where it binds to DNA causing cell cycle arrest (apoptosis). p53C is a part of the extrinsic pathway, but also acts by upregulating Bax and downregulating Bcl-2, which are a part of the intrinsic pathway. Both the intrinsic and extrinsic pathways converge at the executioner caspase cascade, which results in germ cell death.

et al., 2003; Vera et al., 2004). The activated form of Apaf-1 polymerase, lamin and actin, resulting in morphological subsequently binds to initiator (apical) caspase 9 and pro- changes and eventually, apoptosis (Hikim et al., 2003; Vera teolytically activates the caspase cascade via executioner et al., 2005). As previously mentioned, ROS possibly trig- caspases like caspases 3, 6 and 7 (Liu, 2010; Vera et al., 2004). gers this apoptotic pathway directly by causing the release These caspases are involved in the cleavage of various struc- of cytochrome C into the cytosol via altered functions of sig- tural and repair proteins, such as poly(ADP) ribose nalling molecules (Ishii et al., 2005).

Extrinsic apoptotic pathway Conclusion Two pathways have been proposed to explain how germ cells undergo apoptosis extrinsically: the Fas/Fas ligand (FasL) Spermatogenesis involves a complex series of stages that system or p53 system (Ishii et al., 2005; Izu et al., 2004). involve the development of spermatogonia into specialized Fas is a type I transmembrane receptor protein whereas spermatozoa. The developmental processes of the male FasL is a type II transmembrane protein (Miura et al., 2002). gamete may be influenced by various factors, including heat This death receptor pathway requires the ligation of the death stress, causing the production of sperm with lower quality and receptor (Fas) to its ligand (FasL) to be initiated, which causes thus affecting fertility. The factors that contribute to in- Fas to trimerize and recruit Fas-associated death domain creased scrotal temperatures range from lifestyle, occupa- (FADD) through shared death domains (DD) (Hikim et al., 2003; tional, environmental to pathophysiological. Many of these Vera et al., 2004). Activated DD subsequently triggers the factors can hardly be avoided altogether. Testicular thermo- caspase cascade when Fas/FADD complex binds to initiator/ regulation is therefore of great importance to ensure the pro- apical caspases 8 or 10 with its N-terminal death effector duction of viable spermatozoa and to maintain fertility. domain (DED), and this results in the activation of executioner/ However, failure to regulate scrotal temperatures or expo- effector caspases 3, 6 and 7 (Hikim et al., 2003; Miura et al., sure to high temperatures result in testicular heat stress. 2002; Vera et al., 2004). Hikim et al. (2003) and Vera et al. Sperm cells are vulnerable to heat stress and respond by un- (2004) conducted studies on FasL-defective gld (general- dergoing apoptosis (germ cells) and DNA damage (both germ ized lymphoproliferation disease), or Fas-defective lprcg cells and epididymal sperm). Heat stress also modifies gene (lymphoproliferation complementing gld) mice, or both, and expression in the testis that could impair the regular sper- proved that heat stress-induced apoptosis still occurred. These matogenic processes. The consequences of testicular heat results suggest that the Fas/FasL pathway is not necessary for stress to the male germ cell are presented in Figure 2. apoptosis, and that another pathway involving p53 was also Consequences of heat stress on germ cells, however, are a key player in cell death. not thoroughly understood. This warrants further genetic The tumour suppressor p53 is a transcription factor that studies to shed more light on pathways that regulate heat increases the expression of pro-apoptotic genes when exposed stress responses of male germ cells and discover new genes to heat stress and therefore regulates the cell cycle (Miura that may be involved. Understanding the molecular mecha- et al., 2002). In hyperthermic conditions, p53 is relocalized nisms of testicular heat stress would aid in developing tar- from the nuclear envelope into the nucleus, where it binds geted male infertility therapies and contraception. Meanwhile, to DNA and causes cell cycle arrest or apoptosis (Yin et al., both fertile and infertile men who are looking to start or con- 1997a). Yin et al. (1997a) showed that apoptosis levels were tinue their family may do well to refrain from as many dif- lower and delayed, and the amount of DNA damage was higher, ferent types of factors that induce heat stress as possible, so in mice lacking the p53 gene post-heat exposure. This implies that harmful effects of hyperthermia on sperm quality can be that the p53 pathway is not the sole mechanism by which ex- minimized. trinsic apoptosis is carried out (Yin et al., 1998b). It is, therefore, conceivable that both the Fas/FasL and p53 pathways work together to cause apoptosis. Miura et al. (2002) found that the amount of Fas dramatically increased within 1 day of heat exposure whereas p53 was only up- regulated after 3 days, thus suggesting that early apoptosis is Fas/FasL-mediated whereas late apoptosis is carried out via the p53 pathway. Conversely, Yin et al. (1998b) produced conflicting results, which indicated that p53 was in fact responsible for early apoptosis whereas Fas/FasL was involved in the later stages (Yin et al., 1998a). Further experiments are required to determine which system is responsible for which phase of apoptosis, but it is safe to con- clude that both play an important role in the response to heat stress.

Crosstalk between various pathways Furthermore, an extensive degree of crosstalk occurs among the various pathways (Liu, 2010; Vera et al., 2004). For instance, p53 up-regulates the activity of pro-apoptotic genes Figure 2 Autophagy and apoptosis: response of the male germ like Bax by promoting its insertion into the mitochondrial mem- cell to testicular heat stress. Proposed pathways and the re- brane, and down-regulates anti-apoptotic genes like Bcl-2, sponses that lead to apoptosis. When the testicles are under heat both of which are mediators of the intrinsic apoptotic pathway stress, germ cells undergo apoptosis via the intrinsic or extrin- (Miura et al., 2002). Intrinsic and extrinsic pathways also con- sic mechanism. Along with, or as a back up to, apoptosis, au- verge on the downstream effector/executioner caspases 3, tophagy is also responsible for germ cell death. Apoptosis can 6and7(Liu, 2010). As such, the mechanism of apoptosis is occur through several means, such as stress response, DNA damage complex and can be carried out via different pathways, ul- and changes in gene expression, all of which result in impaired timately resulting in germ cell death. DNA repair that lead to germ cell death.

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